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Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration
KTH, School of Biotechnology (BIO), Biochemistry.
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2010 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 46, no 30, 5569-5571 p.Article in journal (Refereed) Published
Abstract [en]

Enantiopure chiral amines synthesis using omega-transaminases is hindered by an unfavourable equilibrium, but when using isopropylamine as the amine donor the equilibrium can be completely displaced by using a specific dehydrogenase in situ for removal of formed acetone.

Place, publisher, year, edition, pages
2010. Vol. 46, no 30, 5569-5571 p.
Keyword [en]
OPTICALLY-ACTIVE AMINES, ASYMMETRIC-SYNTHESIS, OMEGA-TRANSAMINASES, CHIRAL AMINES
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-12935DOI: 10.1039/c0cc00050gISI: 000280145000048OAI: oai:DiVA.org:kth-12935DiVA: diva2:319710
Note
QC 20100519Available from: 2010-05-19 Created: 2010-05-19 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Tools in biocatalysis: enzyme immobilisation on silica and synthesis of enantiopure amines
Open this publication in new window or tab >>Tools in biocatalysis: enzyme immobilisation on silica and synthesis of enantiopure amines
2010 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents two techniques in the field of biocatalysis:

An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol.

An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines.

Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development.

 

Abstract [sv]

I denna avhandling presenteras två tekniker inom ämnet biokatalys:

En metod för immobilisering av His6-enzym på modifierad kiseloxid, och användning av detta konstrukt för kemiska synteser i vatten och organiska lösningsmedel. Detta system fungerar även som en snabb extraherings- och immobiliseringsmetod.

Ett jämviksförskjutningssystem som möjliggör fullständig omsätt-ning i reaktioner med ω-transaminaser där isopropylamin är amino-donator, en syntesväg för tillverkning av farmakologiskt intressanta kirala aminer.

Biokatalys förutspås att bli en ovärderlig teknologi i en miljömässigt hållbar kemisk industri, i vilken varje ny metod är en liten men dock viktig del. Detta arbete är menat som en del i denna utveckling.

Publisher
27 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:11
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-12936 (URN)
Presentation
2010-05-28, 15:00 (English)
Opponent
Supervisors
Note
QC 20100519Available from: 2010-05-19 Created: 2010-05-19 Last updated: 2011-11-29Bibliographically approved
2. ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
Open this publication in new window or tab >>ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biocatalysis offers an alternative to classic chemistry by using enzymes, the protein catalysts of Nature, for production of fine chemicals. Evolution has created enzymes capable of catalysis at moderate temperature of a specific reaction in the presence of a plethora of compounds in the aqueous cell environment. The focal point of biocatalysis is to utilise these traits in vitro, for creation of valuable molecules.

The ω-transaminase is an enzyme capable of producing chiral amines, compounds used to great extent in pharmaceuticals. Much effort has in recent years been invested in the research and engineering of this enzyme type since the catalysed reaction offers an advantageous alternative to classical techniques. Nevertheless, there is a need for method development, adaptation of the enzyme and increased understanding of the catalytic mechanism for feasibility as an effective biocatalyst for unnatural substrates. This thesis addresses a chosen set of obstacles as a contribution to meeting the demands at hand. ω-Transaminase from Chromobacterium violaceum and Arthrobacter citreus was used.

Many homologous ω-transaminases are available, which are also subject to engineering where variants are produced. To accurately compare their kinetic constants an active site quantification method is required but has not been available. Here such a method is presented (Paper 1) which encompasses a virtually irreversible half transamination reaction.

In stereoselective synthesis the ω-transaminase catalysed equilibrium reaction inherently results in incomplete conversion. An equilibrium displacement system is presented (Paper II) where isopropylamine is the amino donor for transamination of acetophenone and derivatives thereof, coupled to an enzymatic cascade reaction.

For many unnatural substrates the specificity and enantiospecificity is insufficient. Rationally redesigned variants were produced with improved properties for chosen substrates (Paper III and IV). The catalytic contributions of field and resonance of a variant compared to the wild type were investigated (Paper IV) for increased knowledge of the mechanism.

For rational redesign of an enzyme the three-dimensional structure is required, of which only a few are available for the ω-transaminases. X-ray crystallographic structures of the holo and apo form of Chromobacterium violaceum ω-transaminase were made (Paper V) which revealed significant structural rearrangements upon coenzyme binding which may be of consequence for future engineering.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. x, 57 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:13
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-92516 (URN)978-91-7501-242-1 (ISBN)
Public defence
2012-04-20, FR4 (Oscar Kleins Auditorium) AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20120402Available from: 2012-04-02 Created: 2012-04-02 Last updated: 2012-04-02Bibliographically approved

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